Use of pramipexole to treat amyotrophic lateral sclerosis

ABSTRACT

The present invention is directed to compositions comprising pramipexole and the use of such compositions to treat neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). As shown in FIG.  6 B the mean +/− SEM serum 2,3-DHBA levels for the 12 ALS participants decreased significantly after pramipexole treatment.

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) to provisionalpatent application No. 60/339,383 filed Dec. 11, 2001 and Ser. No.60/347,371 filed Jan. 11, 2002, the disclosures of which areincorporated herein.

U.S. Government Rights

This invention was made with United States Government support underGrant Nos. NS35325, AG14373, NS39788 and NS39005 awarded by NationalInstitutes of Health. The United States Government has certain rights inthe invention.

FIELD OF THE INVENTION

The present invention relates to the use of pramipexole(2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole) to treatneurodegenerative diseases. More particularly the invention is directedto the use of the substantially pure sterioisomer R(+)2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole and thepharmacologically acceptable salts thereof as a neuroprotective agent totreat neurodegenerative diseases.

BACKGROUND OF THE INVENTION

Neurodegenerative diseases (NDD) such as Alzheimer's disease (AD) andParkinson's disease (PD) arise from the accelerated loss of certainpopulations of neurons in the brain. Parkinson's (PD) and Alzheimer's(AD) diseases usually appear sporadically without any obvious Mendelianinheritance patterns, but may show maternal biases. Although rare oruncommon inherited forms of adult NDD exist, the relevance ofpathogenesis in these autosomal genetic variants to the much morecommonly occurring sporadic forms is a subject of intense debate.

Accumulating evidence provides compelling support that a primaryetiologic component of sporadic adult NDD relates to mitochondrialdysfunction and the resulting increased cellular oxidative stress. PDand AD brains and non-CNS tissues show reductions in mitochondrialelectron transport chain (ETC) activity. When selectively amplified incytoplasmic hybrid (“cybrid”) cell models, mitochondrial genes from PD(Swerdlow, et al, Exp Neurol 153:135–42, 1998) and AD (Swerdlow, et al,Exp Neurol 153:135–42 1997) subjects recapitulate the ETC deficits,produce increased oxidative stress and a variety of other importantmitochondrial and cellular dysfunctions. The combined weight of evidencefrom tissue studies and cybrid models of sporadic PD and AD suggeststhat relief of oxidative stress, by agents capable of scavenging oxygenfree radicals and protecting cells from mitochondrially generated celldeath, be considered as a primary characteristic of compounds developedas neuroprotective agents for these diseases (Beal, Exp Neurol153:135–42, 2000).

Oxidative stress has also been associated with the fatalneurodegenerative disorder amyotrophic lateral sclerosis (ALS). ALS,also known as Lou Gehrig's disease, is a progressive, fatalneurodegenerative disorder involving the motor neurons of the cortex,brain stem, and spinal cord. It is a degenerative disease of upper andlower motor neurons that produces progressive weakness of voluntarymuscles, with eventual death. The onset of disease is usually in thefourth or fifth decade of life, and affected individuals succumb within2 to 5 years of disease onset. ALS occurs in both sporadic and familialforms.

About 10% of all ALS patients are familial cases, of which 20% havemutations in the superoxide dismutase 1 (SOD 1) gene (formerly known asCu,Zn-SOD), suggesting that an abnormally functioning Cu,Zn-SOD enzymemay play a pivotal role in the pathogenesis and progression of familialamyotrophic lateral sclerosis (FALS). It is believed that the increasedgeneration of oxygen free radicals, especially hydroxyl radicals, bymutant SOD1, to be the initiating factor that results in the sequence ofevents leading to motor neuron death in FALS. This hypothesis issupported by recent reports that transfection of neuronal precursorcells with mutant SOD1 results in increased production of hydroxylradicals and enhanced rate of cell death by apoptosis. Furthermore,applicants believe that oxidative stress is responsible motor neurondeath in sporadic forms of ALS as well.

Recent research has revealed that a likely inciting event in thepremature neuronal death that is associated with ALS is the presence ofmutated mitochondrial genes (mitochondrial DNA, mtDNA). These mtDNAmutations lead to abnormalities in functioning of energy productionpathways in mitochondria, resulting in an excessive generation ofdamaging oxygen derivatives known as “reactive oxygen species” (ROS),including entities called “oxygen free radicals.” When ROS productionexceeds the capacity of cellular mechanisms to remove/inactivate ROS,the condition known as “oxidative stress” exists.

Oxidative stress can damage many cellular components. Work by theinventor has shown that a critical cell component damaged by oxidativestress in cell models of AD and PD is a particular mitochondrial proteincomplex known as the “mitochondrial transition pore complex” (MTPC).Normal activity of the MTPC is essential for the maintenance of abioelectric potential (ΔΨ) across mitochondrial membranes, which in turnis used for mitochondrial synthesis of energy storage chemicals such asATP. Loss of ΔΨ results in depolarization of mitochondria and initiatesa cascade of biochemical reactions which ultimately result in cell deathby a mechanism known as “programmed cell death” or “apoptosis.”Apoptosis mechanisms have been observed not only in AD and PD, but alsoin other NDD such as amyotrophic lateral sclerosis (ALS) andHuntington's disease.

Accordingly, one strategy for treating these various neurodegenerativediseases involves the administration of a neuroprotective agent.Effective neuroprotective agents for these debilitating and fatalillnesses should not only be effective in cell culture and animal modelsof these diseases, but must be tolerated chronically in high enoughdoses to achieve therapeutic levels in nervous tissues. Ideally suchagents would also target cellular components involved in control of celldeath pathways and interrupt disease pathophysiology.

In accordance with one embodiment of the present invention a method isprovided for treating a neurodegenerative disease such as ALS. Themethod comprises the steps of administering pramipexole to theindividual in an amount effective to reduce oxidative stress in thatindividual.

Pramipexole (PPX, 2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole)exists as two sterioisomers:

The S(−) enantiomer is a potent agonist at D2 family dopamine receptorsand is extensively used in the symptomatic management of PD. Thesynthesis, formulation and administration of pramipexole is described inU.S. Pat. Nos. 4,843,086, 4,886,812 and 5,112,842, the disclosures ofwhich are incorporated herein. S (−) PPX has been shown by severalgroups to be neuroprotective in cellular and animal models of increasedoxidative stress, including MPTP toxicity to dopamine neurons (see U.S.Pat. Nos. 560,420 and 6,156,777, the disclosures of which areincorporated herein). S(−) PPX reduces oxidative stress produced by theparkinsonian neurotoxin and ETC complex I inhibitor methylpyridinium(MPP+) both in vitro and in vivo and can block opening of themitochondrial transition pore (MTP) induced by MPP+ and other stimuli(Cassarino, et al, 1998). The lipophilic cationic structure of PPX issuggestive of the possibility that concentration into mitochondriaacross ΔΨ_(M), in combination with its low reduction potential (320 mV),may account for these desirable neuroprotective properties.

Dosing with S(−) PPX is limited in humans by its potent dopamine agonistproperties and will restrict achievable brain drug levels. Because theR(+) enantiomer of PPX has very little dopamine agonist activity(Schneider and Mierau, J Med Chem 30:494–498,1987) but may retain thedesirable molecular/antioxidant properties of S(−) PPX, this compound issuggested herein as having utility as an effective inhibitor of theactivation of cell death cascades and loss of viability that occurs inneurodegenerative diseases.

SUMMARY OF THE INVENTION

The present invention provides methods for preventing and/or delayingsymptoms, or alleviating symptoms relating to a wide variety ofneurodegenerative diseases. More particularly, the invention is directedto compositions comprising pramipexole and methods of using suchcompositions to treat amyotrophic lateral sclerosis (ALS).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A represents a time course of MPP+-induced release of cytochrome Cand FIG. 1B represents a time course of MPP+-induced activation ofcaspase 3. SH-SY5Y cells were incubated with 5 mM MPP+ for varying timesand harvested. In FIG. 1A, cells were homogenized in isotonic sucrose,centrifuged, and 100 μg of supernatant protein electrophoresed usingSDS-PAGE, transferred to nylon membranes and immunostained forcytochrome C with enhanced chemiluminescence detection. The mostlefthand band (designated “Ohr” on the x axis) corresponds tomitochondrial cytochrome C at time “0”, and the other bands representelectrophoresed cytoplasmic protein immunostained for cytochrome C.Positions of MW markers are shown on the y axis. Other batches of cellswere assayed for caspase 3 using a commercial system, according tomanufacturer's instructions (Biomol). Caspase assays shown in FIG. 1Bare the results of 3–4 independent experiments. *p<0.05 compared toactivity at 0.25 hrs.

FIG. 2 represents data showing the inhibition of MPP+-induced caspase 3activation by PPX, bongkreckic acid and aristolochic acid. SH-SY5Y cellswere incubated with 5 mM MPP+ for 24 hours in the absence of or presenceof 1 mM S(−) PPX, 1 mM R(+) PPX, 250 μM bongkreckic acid (BKA) or 25 μMaristolochic acid (ARA) or combinations thereof. Cells were then assayedfor caspase 3 activity. Shown in FIG. 2 are the mean +/−SEM for 4–5independent experiments. Lanes A-F represent caspase 3 activation bycells incubated with the following compounds: A, control (no compoundsadded); B, MPP+; C, MPP+/BKA; D, MPP+/S(−) PPX; E, MPP+/R(+) PPX; F,MPP+/ARA. For lanes B, p<0.05 compared to control; and for lanes C—F,p<0.05 compared to MPP+treated cells (lane B).

FIG. 3 represents data showing the inhibition of MPP+-induced caspase 9activation by PPX, bongkreckic acid and aristolochic acid. SH-SY5Y cellswere incubated with 5 mM MPP+ for 4 or 8 hours in the absence of orpresence of 1 mM S(−) PPX, 1 mM R(+) PPX, 250 μM bongkreckic acid (BKA)or 25 μM aristolochic acid (ARA). Cells were then assayed for caspase 9activity. Shown in FIG. 3 are the mean +/−SEM for 3–4 independentexperiments. Lanes A–I represent caspase 9 activation by cells incubatedwith the following compounds: A, control (no compounds added); B, 15 minMPP+; C, 4 hr MPP+; D, 4 hr MPP+/PPX; E, 8 hr MPP+; F, 8 hr MPP+/BKA; G,8 hr MPP+/S(−) PPX; H, 8 hr MPP+/R(+) PPX; I, 8 hr MPP+/ARA. For lanes Cand E, p<0.01 compared to control; and for lanes F, G, H, and I p<0.05compared to control.

FIGS. 4A & 4B. Inhibition of MPP+-induced cell death by PPX enantiomers.SH-SY5Y cells were incubated with 5 mM MPP+ for 24 hours in the absenceof or presence of increasing concentrations of S(−) PPX (FIG. 4A) orR(+) PPX (FIG. 4B). Lanes A–F represent cells treated with 0 nM, 3 nM,30 nM, 300 nM, 3 uM, and 30 um of PPX, respectively. The cells were thenincubated with calcein-AM, washed and read on a fluorescent platereader. The number of independent experiments is indicated on each bar,and the P values by t-test for comparison to no PPX (MPP+ only) areshown above each bar. In the presence of MPP+, 3 nM, R(+) PPX causedgreater calcein retention than S(−) PPX (p=0.035), and 3 μM, S(−) PPXcaused greater calcein retention than R(+) PPX (p=0.027).

FIG. 5 is a graphic representation of the time course of changes inserum 2,3-DHBA levels in ALS and control (CTL) subjects afteradministration of 1.3 grams of aspirin. FIG. 5A shows the means +/−SEMfor changes in serum 2,3-DHBA concentration; FIG. 5B shows the ratio ofserum 2,3-DHBA concentration to salicylate concentration; and FIG. 5Cshows the serum salicylate concentration over the time course.

FIG. 6 is a graphic representation of the effect of pramipexole on serum2,3-DHBA concentration. FIG. 6A indicates the DHBA concentrations inindividual subjects both pre and post pramipexole treatment. Subjects 2,3, 7 and 12 were non-ambulatory. Subjects 3 and 7 were ventilatordependent. FIG. 6B provides the mean +/−SEM serum levels of 2,3-DHBA preand post pramipexole treatment. FIG. 6C provides the mean +/−SEM levelsof serum of 2,3-DHBA concentration/salicylate pre and post pramipexoletreatment.

FIG. 7. Mice were adminstered R(+) PPX in their drinking water and thentreated with a neurotoxin (N-methyl-4-pheny-1,2,3,6-tetrahydropyridine,MPTP) which increases oxidative stress in the brain and forebrain2,3-DHBA levels were determined.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

In describing and claiming the invention, the following terminology willbe used in accordance with the definitions set forth below.

As used herein, the term “purified” and like terms relate to theisolation of a molecule or compound in a form that is substantially freeof contaminants normally associated with the molecule or compound in anative or natural environment.

As used herein, the term “treating” includes prophylaxis of the specificdisorder or condition, or alleviation of the symptoms associated with aspecific disorder or condition and/or preventing or eliminating saidsymptoms.

As used herein, an “effective amount” means an amount sufficient toproduce a selected effect. For example, effective amount of ofpramipexole or derivative thereof encompasses an amount that willinhibit the generation of or decrease the levels of reactive oxygenspecies present in an individual.

As used herein, the term “halogen” means Cl, Br, F, and I. Especiallypreferred halogens include Cl, Br, and F. The term “haloalkyl” as usedherein refers to a C₁–C₄ alkyl radical bearing at least one halogensubstituent, for example, chloromethyl, fluoroethyl or trifluoromethyland the like.

The term “C₁–C_(n) alkyl” wherein n is an integer, as used herein,represents a branched or linear alkyl group having from one to thespecified number of carbon atoms. Typically C₁–C₆ alkyl groups include,but are not limited to, methyl, ethyl, n-propyl, iso-propyl, butyl,iso-butyl, sec-butyl, tert-butyl, pentyl, hexyl and the like.

The term “C₂–C_(n) alkenyl” wherein n is an integer, as used herein,represents an olefinically unsaturated branched or linear group havingfrom 2 to the specified number of carbon atoms and at least one doublebond. Examples of such groups include, but are not limited to,1-propenyl, 2-propenyl, 1,3-butadienyl, 1-butenyl, hexenyl, pentenyl,and the like.

The term “C₂–C_(n) alkynyl” wherein n is an integer refers to anunsaturated branched or linear group having from 2 to the specifiednumber of carbon atoms and at least one triple bond. Examples of suchgroups include, but are not limited to, 1-propynyl, 2-propynyl,1-butynyl, 2-butynyl, 1-pentynyl, and the like.

The term “C₃–C_(n) cycloalkyl” wherein n=8, represents cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

As used herein the term “aryl” refers to a mono- or bicyclic carbocyclicring system having one or two aromatic rings including, but not limitedto, phenyl, benzyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl, andthe like. The term (C₅–C₈ alkyl)aryl refers to any aryl group which isattached to the parent moiety via the alkyl group.

The term “heterocyclic group” refers to a mono- or bicyclic carbocyclicring system containing from one to three heteroatoms wherein theheteroatoms are selected from the group consisting of oxygen, sulfur,and nitrogen.

As used herein the term “heteroaryl” refers to a mono- or bicycliccarbocyclic ring system having one or two aromatic rings containing fromone to three heteroatoms and includes, but is not limited to, furyl,thienyl, pyridyl and the like.

The term “bicyclic” represents either an unsaturated or saturated stable7- to 12-membered bridged or fused bicyclic carbon ring. The bicyclicring may be attached at any carbon atom which affords a stablestructure. The term includes, but is not limited to, naphthyl,dicyclohexyl, dicyclohexenyl, and the like.

The compounds encompassed by the present invention include compoundsthat contain one or more asymmetric centers in the molecule. Inaccordance with the present invention a structure that does notdesignate the stereochemistry is to be understood as embracing all thevarious optical isomers, as well as racemic mixtures thereof.

As used herein, the term neuroprotective agent refers to an agent thatprevents or slows the progression of neuronal degeneration and/orprevents neuronal cell death.

The Invention

The present invention is directed to the use of tetrahydrobenthiazolesto treat neurodegenerative diseases, including ALS. More particularly,the tetrahydrobenthiazoles of the present invention have the generalstructure:

wherein R₁, R₂, R₃, and R₄, are independently selected from the groupconsisting of H, C₁–C₃ alkyl and C₁–C₃ alkene. In one preferredembodiment the NR₃R₄ group is in the 6-position. In another embodimentR₁, R₂ and R₄, are H, R₃ is C₁–C₃ alkyl, and the NR₃R₄ group is in the6-position. In one embodiment the compound has the general structure offormula I, wherein R₁ and R₂ are each H and R₃, and R₄ are H or C₁–C₃alkyl.

In accordance with one embodiment, the present invention is directed toa method of treating ALS. The method comprises administering to apatient a compound having the general structure:

wherein R₁ and R₂ are H, and R₃, and R₄, are H, or C₁–C₃ alkyl. In onepreferred embodiment R₁, R₂ and R₄, are H, and R₃ is propyl. In anotherembodiment, the composition comprises pramipexole wherein pramipexolecomponent consists essentially of one of the two sterioisomers ofpramipexole (eitherR(+)-2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole orS(−)-2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole). In oneembodiment the active agent of the composition consists of thepramipexole sterioisomer,R(+)-2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazoledihydrochloride, or other pharmacologically acceptable salts,substantially free of its S(−) enantiomer. In one embodiment acomposition is provided wherein greater than 80% of the pramipexolecompounds present in the composition are in the R(+) conformation, andmore preferably greater than 90% or greater than 95% of the pramipexolecompounds are in the R(+) conformation. In one embodiment a compositioncomprising pramipexole is provided wherein greater than 99% of thepramipexole compounds are in the R(+) conformation.

In one embodiment a composition is provided that comprises an activeagent consisting essentially ofR(+)-2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazoledihydrochloride, or other pharmacologically acceptable salts thereof,and a phamaceutically acceptable carrier. This composition can beadministered orally on a chronic basis for preventing neural cell lossin NDD (and more particularly reducing oxidative stress in ALSpatients), or it can be formulated and administered intravenously forprevention of neural cell loss in acute brain injury.

The neuroprotective effect of the compositions of present inventionderives at least in part from the active compound's ability to preventneural cell death by at least one of three mechanisms. First, thepresent tetrahydrobenzthiazoles are capable of reducing the formation ofROS (both in vivo in rat brain and in vitro in cells with impairedmitochondrial energy production) induced with neurotoxins that can mimicPD. In this manner the tetrahydrobenzthiazoles function as “free radicalscavengers.” Second, the tetrahydrobenzthiazoles can partially restorethe reduced ΔΨ that is correlated with AD and PD mitochondria. Third,tetrahydrobenzthiazoles can block the apoptotic cell death pathwayswhich are produced by pharmacological models of AD and PD mitochondrialimpairment.

In accordance with one embodiment a method is provided for reducingoxidative stress in an ALS patient. A reduction in oxidative stress, forthe purposes of the present invention, is intended to include anyreduction in the level of reactive oxygen species present in thepatient, including for example, decreased serum ROS levels as detectedby the conversion of salicylate to 2,3 DHBA. The method comprisesadministering an effective amount of a tetrahydrobenzthiazole having thegeneral formula:

wherein R₁, R₂ and R₄, are H, and R₃ is C₁–C₃ alkyl. In one embodiment,the tetrahydrobenzthiazole isR(+)-2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole orS(−)-2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole or a racemicmixture of the R(+) and S(−) sterioisomers. The amount oftetrahydrobenzthiazole administered to treat ALS will vary based onroute of administration, as well as additional factors including theage, weight, general state of health, severity of the symptoms,frequency of the treatment and whether additional pharmaceuticalsaccompany the treatment. The dosage amount of the active compound to beadministered is easily determined by routine procedures known to thoseof ordinary skill in the art.

Alternatively, the present invention provides a method for enhancing thebioelectric potential (Ψ) across mitochondrial membranes of cells withimpaired mitochondrial energy production. The method comprises the stepsof contacting cells having impaired mitochondrial energy production witha composition comprising a pramipexole active agent and aphamaceutically acceptable carrier. In one embodiment the pramipexoleactive agent consists essentially of the R(+)2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole sterioisomer andthe pharmacologically acceptable salts thereof.

A number of central nervous system diseases and conditions result inneuronal damage and each of these conditions can be treated with thetetrahydrobenzthiazole compositions of the present invention. Conditionswhich can lead to nerve damage include: Primary neurogenerative disease;Huntington's Chorea; Stroke and other hypoxic or ischemic processes;neurotrauma; metabolically induced neurological damage; sequelae fromcerebral seizures; hemorrhagic stroke; secondary neurodegenerativedisease (metabolic or toxic); Parkinson's disease, Alzheimer's disease,Senile Dementia of Alzheimer's Type (SDAT); age associated cognitivedysfunctions; or vascular dementia, multi-infarct dementia, Lewy bodydementia, or neurogenerative dementia.

The invention is also safe to administer to humans. S(−) pramipexole,which is a potent dopamine agonist approved for the treatment of PDsymptoms is the enantiomer of R(+) pramipexole. However, R(+)pramipexole lacks pharmacological dopamine activity. Accordingly, R(+)2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole and thepharmacologically acceptable salts thereof can be administered in muchlarger doses than S(−) pramipexole and can achieve brain levels capableof providing neuroprotection. In accordance with one embodiment ALS istreated by administering either R(+) or R(−) pramipexole, however theadministration of R(+)2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole is preferredbecause much higher doses can be given. As indicated in Example 1, theS(−) and R(+) isomers are approximately equipotent in reducing oxidativestress. However the use of the R(+) isomer allows one to administerhigher doses and thus achieve greater reduction in toxic oxygen freeradicals. Accordingly, in one embodiment a method of reducing neuralcell death in patients with amyotrophic lateral sclerosis (ALS) isprovided, wherein the patient is administered a pharmaceuticalcomposition comprising a compound of the general structure:

The synthesis of Pramipexole is described in European Patent 186 087 andits counterpart, U.S. Pat. No. 4,886,812, the disclosure of which isincorporated herein.

In one embodiment, pramipexole, and more preferablyR(+)-2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole is compoundedwith binding agents to yield tablets for oral administration, or withsubstances known to the art to yield a transdermal patch (“skin patch”)for continuous delivery. Alternatively, pramipexole can be formulatedwith the necessary stabilizing agents to produce a solution that can beadministered parenterally (ie, intravenously, intramuscularly,subcutaneously). The oral and/or transdermal preparations of theinvention are used to reduce neural cell death in patients with NDD (ieAlzheimer's disease, Parkinson's disease, Huntington's disease,amyotrophic lateral sclerosis). The parenteral formulations of theinvention are used to reduce neural cell death in patients with acutebrain injury (ie. stroke, sub-arachnoid hemorrhage, hypoxic-ischemicbrain injury, status epilepticus, traumatic brain injury, hypoglycemicbrain injury).

The use of R(+) pramipexole to treat NDD, by virtue of its being arelatively inactive stereoisomer of the dopamine agonist S(−)pramipexole (Mirapex, Pharmacia and Upjohn), solves an important problemassociated with the use of S(−) pramipexole as a dopamine agonist.Dosing of Mirapex is limited by dopaminergic side effects on bloodpressure and mentation.R(+)-2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole has 1% orless of the potency to produce the side effects that result from the useof S(−) pramipexole. Thus, the present invention can be administeredmore safely to AD patients, who are typically intolerant of even smalldoses of dopamine agonist medication. Also the present invention can beadministered intravenously in much larger doses than S(−) pramipexole.Thus, it can safely be used in conditions such as stroke, where loweringof blood pressure can be detrimental.

In one embodiment a method for treating a patient having aneurodegenerative disease is provided, that simultaneously reduces therisk of dopaminergic side effects. The method comprises the step ofadministering a composition comprising a pramipexole active agent and aphamaceutically acceptable carrier, wherein the pramipexole active agentconsists essentially of the R(+)2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole sterioisomer andthe pharmacologically acceptable salts thereof. In one embodiment theneurodegenerative disease to be treated is selected from the groupconsisting of ALS, Alzheimer's disease and Parkinson's disease, and thecomposition is administered at a dosage of about 10 mg to about 500 mgper day of R(+) 2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole orthe pharmacologically acceptable salts thereof.

The dosage to be used is, of course, dependent on the specific disorderto be treated, as well as additional factors including the age, weight,general state of health, severity of the symptoms, frequency of thetreatment and whether additional pharmaceuticals accompany thetreatment. The amounts of the individual active compounds are easilydetermined by routine procedures known to those of ordinary skill in theart. For example, the tetrahydrobenzthiazoles of the present inventioncan be administered orally to humans with NDD in daily total dosesbetween 10 mg and 500 mg. Alternatively, the tetrahydrobenzthiazoles canbe administered parenterally to humans with acute brain injury in singledoses between 10 mg and 100 mg, and/or by continuous intravenousinfusions between 10 mg/day and 500 mg/day.

In one preferred embodiment, R(+)2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole (or apharmacologically acceptable salts thereof) is administered to a patientsuffering from a neurogenerative disease such as ALS to treat thedisease. As used herein, the term “treating” includes alleviation of thesymptoms associated with a specific disorder or condition and/orpreventing or eliminating said symptoms. More particularly, acomposition comprising a pramipexole active agent and a pharmaceuticallyacceptable carrier is administered to the patient to prevent orsubstantially reduce neural cell death, wherein the pramipexole activeagent consists essentially of the R(+)2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole (and thepharmacologically acceptable salts thereof) sterioisomer of pramipexole.The synthesis, formulation and administration of pramipexole isdescribed in U.S. Pat. Nos. 4,843,086; 4,886,812; and 5,112,842; whichare incorporated by reference herein.

Hydroxyl radical generation in the tissues of an individual results inan increase in the conversion of salicylate to 2,3 dihydroxybenzoic acid(DHBA) (Floyd et al. (1984) J. Biochem. Biophys. Methods 10:221–235;Hall et al. (1993) J. Neurochem. 60:588–594). Thus the accumulation of2,3 DHBA in the serum of individuals can be used as an indicator oflevel of oxidative stress suffered by that individual. The effect ofR(+) 2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole or S(−)2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole on 2,3dihydroxybenzoic acid (DHBA) serum levels was investigated. As is shownin FIGS. 5 and 6, individual serum levels of 2,3 DHBA decreased in ALSpatients after treatment with S(−)2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole. These datademonstrate that treatment with pramipexole lowers oxidative stress invivo in ALS patients.

Furthermore, as shown in FIG. 7 additional studies were conducted onmice and demonstrate the effectiveness of pramipexole in reducingoxidative stress in vivo. In this study mice were administered R(+) PPXin their drinking water for 8 weeks at 3 different daily doses, and thenwere given a neurotoxin (N-methyl-4-pheny-1,2,3,6-tetrahydropyridine,MPTP) which increases oxidative stress in the brain. Brain tissue wasthen analyzed for oxygen free radical production. The data show that the30 mg/kg/day and 100 mg/kg/day doses resulted in significant reducedfree radical levels in the brain.

Toxicology studies have also been conducted and no evidence of adverseeffects were detected. In particular, an 8 week toxicology study wasperformed in the mice given R(+) PPX in their drinking water. All theirmajor organs were examined pathologically and no lesions were found.While this does not address the issue of effectiveness of R(+) PPX as aneuroprotectant, it does demonstrate the potential safety (and thusfeasibility) of administering very high doses of the drug to humanschronically.

The present invention is also directed to pharmaceutical compositionscomprising the tetrahydrobenzthiazole compounds of the presentinvention. More particularly, the tetrahydrobenzthiazole compounds canbe formulated as pharmaceutical compositions using standardpharmaceutically acceptable carriers, fillers, solublizing agents andstabilizers known to those skilled in the art.

Pharmaceutical compositions comprising the tetrahydrobenzthiazoles areadministered to an individual in need thereof by any number of routesincluding, but not limited to, topical, oral, intravenous,intramuscular, intra-arterial, intramedullary, intrathecal,intraventricular, transdermal, subcutaneous, intraperitoneal,intranasal, enteral, topical, sublingual, or rectal means.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. In accordance with oneembodiment a kit is provided for treating an ALS patient. In thisembodiment the kit comprises one or more of the tetrahydrobenzthiazolesof the present invention, and more particularly the R(+)2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole sterioisomer.

These pharmaceuticals can be packaged in a variety of containers, e.g.,vials, tubes, microtiter well plates, bottles, and the like. Preferably,the kit will also include instructions for use.

EXAMPLE 1

R(+) and S(−) PPX are Effective Inhibitors of Activation of Cell DeathCascades.

Materials and Methods

Cell culture

SH-SY5Y human neuroblastoma cells were obtained from the American TissueCulture Collection (www.atcc.org) and maintained in culture in areplicating state. For caspase assays and cytochrome C release studiesthey were grown in T75 flasks with DMEM/high glucose containing 10%fetal bovine serum, antibiotic/antimicotic (100 IU/ml of penicillin, 100μg/ml of streptomycin sulfate, 0.25 μg/ml of amphotericin B) and 50μg/ml of uridine and 100 μg/ml of pyruvate in a 5% CO₂ atmosphere at 37°C. to approximately full confluence (2×10⁷ cell/flask). They were thenincubated with 5 mM methylpyridinium iodide (MPP+; Sigma;www.sigma-aldrich.com) or 100 μM 25–35 or 35–25 beta amyioid peptides(Bachem; www.bachem.com) for varying times, then harvested. For celldeath studies, the cells were plated into 96 well black-bottom platesand grown for 24 hours in DMEM media before being exposed to toxin.

Caspase Assays

After exposure to MPP+ or beta amyloid peptide, cells were collected inPBS and centrifuged at 450×g for 6 minutes at 4° C. Cell pellets wereresuspended in a hypotonic cell lysis buffer [25 mM HEPES, 5 mM MgCl₂, 5mM EDTA, 1M DTT and protease inhibitor cocktail (Sigma Chemical)] at aconcentration of 2×10⁷ cells/100 μl of lysis buffer. Lysates weresubjected to four cycles of freezing and thawing. Cell lysates were thencentrifuged at 16,000×g for 30 minutes at 4° C. The supernatantfractions were collected, and protein content was measured by Lowryassay (BioRad). 100 μg of protein was used to measure caspase activityin 96 well plates and was assayed in quadruplicate. The activity wasmeasured using the assay buffer and the protocol provided by themanufacturers. (Biomol, caspase 3; Promega, caspases 3 and 9). Caspaseactivity is based on cleavage of synthetic peptide substrates resultingin liberation of colored (p-nitroaniline (p-NA), Biomol) or fluorescent(aminomethylcoumarin (AMC), Promega) chromogens. Only activated caspasesare capable of cleaving these substrates, and chromogen generation iscompletely inhibited when the caspase inhibitor provided with each assaykit is included in the assay. Under the indicated assay conditionslinear rates of generation of chromogen were observed over 2 hours.Chromogen absorbance (p-NA) was measured on an OptiMax plate reader orchromogen fluorescence (AMC) on a SpectraMax Gemini plate reader at zerotime and after 30 minutes of incubation at 37 degrees to estimaterelative caspase activities. Chromogen signal at zero time wassubtracted from the readings at 30 minutes.

Cell Death

Cell death was estimated by measuring loss of calcein retention with the“Live-Dead” assay (Molecular Probes; www.molecularprobes.com) in cellsgrown in 96 well plates and incubated with calcein-AM, according tomanufacturer's instructions. Calcein signals were assayed in aSpectraMax Gemini adjustable fluorescent plate reader (MolecularDevices). Calcein fluorescence from cells preincubated with methanol wassubtracted from all readings as background. Each assay was performedwith 8 wells/condition, which were averaged. 3–8 independent experimentswere performed to evaluate a broad range of concentrations of S(−) andR(+) PPX in this paradigm.

Cytochrome C Western Blot

Cytochrome C was detected by Western blot following polyacrylamideelectrophoresis of 100 μg of cell supernatant protein and transfer tonylon membrane.

The primary antibody was a mouse monoclonal anti-cytochrome C, obtainedfrom Pharmingen and used at 1:10,000 dilution. Detection was performedwith enhanced chemiluminescence (Pierce) and imaged on a BioRad FluorSimaging station.

Drugs

R(+) and S(−) PPX (gifts of Pharmacia Corporation) were obtained astheir dihydrochloride salts and dissolved directly into culture media.Bongkreckic acid, an antagonist of the ATP binding site on the adeninenucleotide translocator, was provided as a solution in 1M NH₄OH.Aristolochic acid (sodium salt), a phospholipase A2 inhibitor, wasobtained from Sigma Chemical Co. In the caspase experiments, drugs wereadded 1 hour before MPP+ or beta amyloid peptide. In the calcein/celldeath experiments, drugs were added 4 hours before MPP+.

Results

Activation of caspases by MPP+ and BA 25–35

FIG. 1B shows the time course of caspase 3 activity during incubation ofSH SY5Y cells with 5 mM MPP+. Increased activity was detectable by 4hours and had increased about 2-fold by 24 hours. FIG. 1A shows theWestern blot result for cytochrome C protein released into cytoplasm.Similar to the biochemical activity curve, cytoplasmic cytochrome C isdetectable in small amounts by 4 hours and increases substantially by 12hours.

FIG. 2 shows that both R(+) and S(−) PPX enantiomers suppressed caspase3 activation during MPP+exposure. MPP+-induced increases in caspase 3activity were also blocked by bongkreckic acid, a specific antagonist ofthe ATP binding site on the inner membrane site of the adeninenucleotide translocator.

Aristolochic acid, a phospholipase A2 inhibitor, previously shown toblock MPP+-induced apoptosis of SH-SY5Y cells (Fall and Bennett, 1998),also prevented increases in caspase 3 activity. Activation of caspasesby MPP+ and BA 25–35 peptide was blocked by PPX enantiomers and agentsactive at the mitochondrial transition pore. S(−) PPX reduced by about70% the increases in caspase 3 activity following incubation with BA25–35 peptide and had no suppressive effect by itself. MPP+ exposurealso increased activity of caspase 9 with a time course similar toactivation of caspase 3 (see FIG. 3). The increases in caspase 9activity were also blocked by S(−) and R(+) PPX, bongkreckic acid andaristolochic acid.

FIG. 4 shows the effects on cellular calcein retention of incubatingSH-SY5Y cells with varying concentrations of R(+) or S(−) PPX prior toexposure to 5 mM MPP+ for 24 hours. Calcein is a fluorescent dye that isretained inside cells as a function of their ability to maintain aplasma membrane potential. MPP+ alone reduced calcein uptake by about60%. Both PPX enantiomers substantially restored calcein uptake at 30 nMlevels, and this protective effect was retained through 30 μM PPX's.

Discussion

This study focused on the use of the neurotoxins MPP+ and 25–35 betaamyloid peptide added to replicating SH-SY5Y neuroblastoma cells as cellculture models for studying potential neuroprotective compounds usefulfor Parkinson's and Alzheimer's diseases, respectively. SH-SY5Y cellsare neoplastic, dividing cells of neuroectodermal origin, not primaryneurons. They are mitotic as a result of a Ras mutation, leading tochronic activation of MAPK/ERK signaling. SH-SY5Y cells are relativelyinsensitive in short-term incubations to MPP+, compared to primaryneurons. Applicants have found that 2.5 and 5 mM, but not 1 mM, MPP+produced apoptotic morphology and DNA pyknotic fragments within 18–24hours. However, longer incubations with lower concentrations of MPP+ maymore closely approximate the in vivo MPTP model of PD in animals, and ithas been reported that longer term exposure to lower MPP+ levels stillcan activate the mitochondrial cell death cascade.

SH-SY5Y cells are also sensitive to beta amyloid peptides. Li, et al(1996) found that serum-starved SH-SY5Y showed extensive DNA nicked endlabeling after 3 days exposure to 100 μM beta amyloid 2S–3S andexhibited a concentration-dependent increase in DNA laddering. Betaamyloid peptide-induced activation of caspases has not apparently beendescribed in SH-SY5Y, but several reports have shown caspase activationsfrom exposure to beta amyloid peptides in various primary neuron lines.These include activation of caspases-2, -3 and -6 by the 25–35 analoguein cerebellar granule cells, and caspase-3 in rat primary corticalneurons. Thus, it is not surprising that caspase-3 activity (DEVDase)was observed in SH(−)SY5Y exposed to 100 μM beta amyloid 25–35 but nocaspase activation was observed after exposure to the reverse 35–25sequence.

The focus of the present experiments was to determine if PPX enantiomerscan prevent activation of caspases and can promote calcein retention asa marker of cell survival in acute toxin exposure, cell culture modelsof AD and PD. Activation of both the “initiator” caspase 9 and“executioner” caspase 3 was blocked by both PPX enantiomers in theMPP+model for PD, and activation of caspase 3 was blocked by S(−) PPX inthe BA 25-3S model for AD. Both PPX enantiomers at nanomolar levelscould promote cell survival in the MPP+ model for PD. Thus, the presentfindings add to the growing body of work describing the neuroprotectiveactions of PPX and suggest potential clinical utility of this family ofcompounds in neurodegenerative diseases.

While this study did not examine the most proximate site of action ofPPX, several finding implicate the mitochondrial transition pore complex(MTPC) in these cell models. First, bongkreckic acid, a selectiveadenine nucleotide translocator antagonist and inhibitor of MTP opening,blocked MPP+ induced activation of both caspases 9 and 3. This would beconsistent with MPP+ bringing about MTP opening either directly orthrough mechanisms that involve oxidative stress. In isolated livermitochondria MPP+ can bring about a classical MTP opening that isincompletely blocked by free radical scavenging enzymes and is morecompletely blocked by S(−) PPX. Neurotoxic 25–35 BA peptide can alsostimulate MTP opening in isolated mitochondria. Both MPP+ and 2S-35 BApeptide increase oxidative stress in brain microdialysis studies in vivoand in neural cell culture in vitro, and S(−) PPX has been shown toreduce MPP+-induced oxidative stress in vitro and in vivo.

EXAMPLE 2

Use of Pramipexole to Treat ALS

Oxidative abnormalities have been identified both in familialamyotrophic lateral sclerosis (FALS) and the more prevalent sporadic ALS(SALS). 2,3-DHBA is a hydroxylated salicylate byproduct that has beenshown to be a reliable in vivo marker of increased free radical activityand is reliably assayed by HPLC. Following the administration of an oralsalicylate load, elevated serum levels of 2, 3-dihydroxybenzoic acid(2,3-DHBA) and DHBA/salicylate were observed in SALS subjects. Asdescribed herein 12 SALS patients were studied to determine the levelsof 2,3-DHBA both before and after treatment with pramipexole.

Methods

Participant Preparation

The present study was conducted in two phases. In the first phase elevendefinite SALS subjects who met Airlie House criteria, and 7 controlswere studied. These participants underwent aspirin loading withsubsequent 2,3-DHBA analysis. After receiving 1.3 grams of aspirin po,blood was drawn 2, 3, and 4 hours later. Serum was separated, frozen andstored at −80 degrees. Aliquots of serum were subsequently coded andblinded for 2,3-DHBA and salicylate assays.

In the second phase, 17 subjects with definte SALS were randomlyselected from a clinic population. The subjects received 1.3 grams ofaspirin po, and blood was drawn 3 hours later. Following acquisition ofthese baseline samples, SALS participants began pramipexole therapy.Dosage escalation was performed similar to PD patients with an attemptmade to reach 1.5 mg t.i.d.-q.i.d. as the final dose, following a 7 weektitration. After each participant was on his/her highest pramipexoledose for three weeks, the aspirin loading study was performed onceagain. Twelve of the original seventeen SALS subjects could complete thepramipexole escalation phase. Of these all but two were able to reach apramipexole dosage of 6 mg per day, and all patients obtained at least adosage of 3 mg per day. Each participant was then offered theopportunity to continue with pramipexole treatment.

Specimen Preparation

Preparation of serum: 0.9 ml of serum at 4° C. was mixed with 0.2 ml of1 M perchloric acid and centrifuged at 15,000 rpm for 10 minutes in arefrigerated microcentrifuge.

Assay for 2,3-DHBA: 20 uL of supernatant were injected in duplicate ontoa C18 “Catecholamine” Adsorbosphere column (Alltech) perfused at 0.6ml/min with buffer consisting of 125 ml/L acetonitrile, 1.5 gn/L ofsodium heptane sulfonate, 3 ml/L triethylamine, 100 mg/L of Na₂ EDTAwith final pH adjusted to 2.8 with phosphoric acid. Detection utilized aCouloChem II flow-through electrochemical detector (ESA, with thefollowing settings: guard cell=+600 mV; E1=−100 mV; E2=+400 mV).2,3-DHBA eluted at 10–10.5 minutes under these conditions.

Assay for salicylate: 50 uL of supernatant were injected in duplicateonto a C8 Kromosil HPLC column (Alltech) perfused at 0.8–1.0 ml/min with70% methanol/30% water/0.5% trifluoroacetic acid. Salicylate eluted at6–7 minutes under these conditions and was detected by ultravioletabsorption at 315 nM.

Results

FIG. 5A shows the time course of increase in 2,3-DHBA serum levels inthe 11 SALS (59.2±12.3 yr) and 7 age matched control (56.7±10.7 yr)subjects studied in the first phase. The SALS patients exhibited a rangefrom symptom onset of 10 to 156 months, representing both acute andchronic stages of this disease. Maximum 2,3 DHBA levels were found inSALS subjects at 3 hours after aspirin dosing, and this time point waschosen for the second phase of the study.

In addition, 2-way ANOVA revealed a difference in production of 2,3-DHBAwhen SALS and control groups were compared across time. This differencewas significant at the p=0.033 level for the two populations; post-hoctesting (Tukey test) did not reveal any significant differences betweenindividual time points.

As an additional comparison, ratios of 2,3-DHBA/salicylate were comparedacross time for the two populations. The ALS group showed an approximate1.5-fold increase in this normalized marker of 2,3-DHBA production at 3hours after administration of aspirin. 2-way ANOVA showed a differencesignificant at the p=0.06 level (FIG. 5B). Serum salicylate levels werenot significantly different over time between the ALS and CTLpopulations (FIG. 5C). Of the original 17 patients who entered thesecond phase of the study, 5 dropped out due to complications from thedisease or inability to tolerate the medication. The remainingparticipants consisted of 8 males and 4 females. The average age was63.2 years. Pramipexole therapy was well tolerated by these ALSsubjects. These participants did not exhibit any evidence of clinicaldementia, nor did they have significant cardiovascular instability.

The patients enrolled in this study represented various clinical stagesof disease progression. Four participants were non-ambulatory, two ofwhich were ventilator-dependant. The other eight were ambulatory at theonset of the study. The average time from the entry into the study andthe final blood draw was 76.6 days with a range of 49–105 days.

Serum levels of 2,3-DHBA were compared pre- and post pramipexoletreatment. Without exception, the individual serum levels of 2,3-DHBAwere reduced (FIG. 6A). FIG. 6B shows the mean+/−SEM for serum 2,3-DHBAconcentration in the ALS subjects before and after achieving stablepramipexole dosing. The mean reduction was about 45% and was significantat the p=0.015 level. Serum salicylate levels (μM) were unchanged bothbefore (18.8+/−7.2, S.D.) and during (20.2+/−5.5, S.D.) pramipexoletreatment. FIG. 6C shows that serum levels of 2,3-DHBA normalized tosalicylate levels for each subject were reduced an average of 59% bypramipexole treatment; t-test showed this difference to be significantat the p=0.010 level.

Discussion

This study revealed that an approximate two-fold increase of in vivooxidative stress was observed in ALS patients after oral aspirinloading, based on an increased production of 2,3-DHBA. The increase in2,3-DHBA was observed at various stages of clinical disease progression,suggesting that this metabolite may serve as a reliable marker ofincreased oxidative stress throughout this disease process, butparticularly in the early stages, when the diagnosis often remainsuncertain. Because of the small population size, no attempt was made tocorrelate the level of 2,3-DHBA production with disease stage.

The present study also revealed that pramipexole therapy, at dosestypically tolerated in Parkinson's disease, reduced in vivo oxidativestress in ALS subjects. Serum from 12 patients before and several weeksafter reaching maximum tolerated doses of pramipexole revealed areduction of about 45% of basal 2,3-DHBA and about 59% of basal 2,3-DHBAnormalized to salicylate level. It is likely that pramipexole broughtabout this reduction in ROS production as a result of its free-radicalscavenging/antioxidant properties. Pramipexole has been shown capable ofreducing ROS production in both SY5Y neuroblastoma cells in vitro andrat striatum in vivo acutely exposed to complex I inhibition withmethylpyridinium (Cassarino et al., J. Neurochem 1998; 71:295–301).Pramipexole also reduces brain ROS production in vivo following infusionof the neurotoxin 6-hydroxydopamine (Ferger et al., Brain Res 2000;883:216–23), inhibits cytochrome C release and reduces brain lipidoxidation in vivo following treatment with the pro-neurotoxin MPTP [16].

Since approximately equivalent neuroprotective actions of pramipexoleare observed in the R(+) and S(−) enantiomers, and since dopamineagonist actions reside primarily in the S(−) enantiomer, theROS-scavenging actions likely have no relationship to dopamine agonistproperties. If this is true, then the R(+) enantiomer of pramipexoleshould be tolerated in much higher doses than the S(−) enantiomer usedin the present study, with the potential for increased antioxidativeactivity in vivo.

Oxidative stressors are well established in sporadic ALS, however theetiology of increased oxidative stress remains obscure. Markers of CNSoxidative stress damage in ALS include increased immunohistochemicalstaining in lumbar spinal cord for lipid peroxidation product, andincreased spinal fluid levels of nitrotyrosine and nitrosylatedmanganese superoxide dismutase. These results are consistent withincreased damage to ALS tissue by ROS that include nitric oxidederivatives.

Animal and cell models of ALS also provide insight into oxidative stressand motor neuronal vulnerability. ALS spinal cord motor neurons havereduced activity of cytochrome C oxidase, (studied histochemically) andsuch reduced electron transport chain function may serve to increaseoxygen free radical production. Spinal cord microdialysis of micecarrying a mutant human SOD1 FALS gene revealed increased production ofROS and levels of malondialdehyde, consistent with that concept.Furthermore, it has been reported that a CuZn-SOD FALS mutationincreased the vulnerability of spinal motor neurons to death fromexcitotoxicity, and the mechanism involved increased oxidative stress.Finally, increased oxidative stress in ALS may be responsible forincreased markers of apoptotic cell death processes observed in ALSspinal cord, and for involvement of caspases in motor neuron death in aFALS mouse model.

Mitochondrial pathology occurs early in the course of experimental motorneuron disease. These structures are not only sensitive to oxidativeinjury, but their dysfunction leads to accelerated free radicalproduction and possible damage to mitochondrial DNA. Visualization ofmitochondrial function in ALS muscle biopsies revealed reduced activityof complex I, and ultrastructural analysis of ALS anterior horn celldendrites revealed aggregated, dark mitochondria. Selective loss ofglial excitatory amino acid transporter-2 (EAAT2) around degeneratinganterior horn cells in ALS may reflect additional protein damage thatcould contribute to increased excitotoxicity.

Oxidative damage in ALS may therefore represent a primaryneurodegenerative process, a secondary epiphenomena of motor neuronmitochondrial pathology, or a combination of both. Cybrid studies withamplification of SALS mitochondrial genes have shown that increasedoxidative stress in SALS can be understood in terms of a primarymitochondrial genetic etiology. How mitochondrial DNA becomes defectivein SALS is not known; both maternally inherited and sporadicallyacquired mechanisms are possible.

The use of salicylate loading and 2,3-DHBA measurements as an estimateof relative in vivo oxidative stress levels has been applied toadult-onset diabetes, liver dysfunction in alcoholics and arthritis.Multiple oxidizing species can contribute to the production of 2,3-DHBA,including hydroxyl radical and peroxynitrite anion. Thus, a particularROS source cannot be assigned for the observed 2,3-DHBA levels, nor canthe tissue source(s) of increased salicylate hydroxylation be defined.

In summary, basal serum levels of 2,3-DHBA, a marker of oxidativestress, were found to be increased in a cohort of SALS subjects, andthese levels were reduced following treatment with pramipexole. As isshown in FIG. 6 individual serum levels of 2,3 DHBA decreased in ALSpatients after treatment with S(−)2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole. In particular,the patients were administered orally a daily dose of 3–6 mg of S(−)2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole for seven weeks.At the end of seven weeks serum samples were taken and the concentrationof 2,3 DHBA was measured and compared to 2,3 DHBA levels in serumsamples taken prior to treatment. These data demonstrate that treatmentwith pramipexole lowers oxidative stress in vivo in ALS patients.

EXAMPLE 3

Effectiveness of Pramipexole in Reducing Mptp Induced Oxidative Stressin vivo

Methods

Male C57BL/6 mice received daily R(+) pramipexole dihydrochloride indrinking water for 8 weeks at doses calculated to provide 0, 10, 30 or100 mg/kg/day. On the day of the study the mice were injected with 30mg/kg s.c. of the neurotoxinN-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to increaseoxidative stress in the brain. One hour later the mice were injectedwith 100 mg/kg i.p. of sodium salicylate. One hour after salicylateinjection the mice were killed and forebrains analyzed for2,3-dihydroxybenzoic acid (2,3-DHBA) content.

As shown in FIG. 7 the results show that 30 and 100 mg/kg/day treatmentwith R(+) pramipexole significantly reduced forebrain oxidative stressproduced by MPTP. Toxicology studies have also been conducted and noevidence of adverse effects were detected. In particular, an 8 weektoxicology study was performed in the mice given R(+) PPX in theirdrinking water. All their major organs were examined pathologically andno lesions were found.

1. A method of treating ALS in a patient in need thereof, comprisingadministering to the patient a pharmaceutical composition comprising apharmaceutically acceptable carrier and an effective amount ofpramipexole, wherein greater than 90% of the pramipexole is R(+)2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole.
 2. The method ofclaim 1 wherein greater than 95% of the pramipexole is R(+)2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole.
 3. The method ofclaim 1 wherein greater than 99% of the pramipexole is R(+)2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole.
 4. The method ofclaim 1 wherein the pramipexole is administered in an amount effectiveas a neuroprotectant.
 5. The method of claim 1 wherein about 3 mg toabout 500 mg of pramipexole is administered daily.
 6. The method ofclaim 1 wherein the pramipexole is R(+)2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole.
 7. The method ofclaim 1 wherein between about 3 mg to about 6 mg is administered daily.8. The method of claim 1 wherein about 30 mg/kg to about 100 mg/kg ofpramipexole is administered per day.
 9. The method of claim 1 whereinthe pramipexole is administered via a route selected from the groupconsisting of transdermal, topical, oral, intravenous, intramuscular,intra-arterial, intramedullary, intrathecal, intraventricular,subcutaneus, intraperitoneal, intranasal, enteral, sublingual, andrectal.
 10. The method of claim 1 wherein said the pramipexole isadministered in a dosage escalation regime.